The pro‐inflammatory signature of lipopolysaccharide in spontaneous contracting embryoid bodies differentiated from mouse embryonic stem cells

Abstract Embryonic stem (ES) cells differentiate towards all three germ layers, including cardiac cells and leukocytes, and may be therefore suitable to model inflammatory reactions in vitro. In the present study, embryoid bodies differentiated from mouse ES cells were treated with increasing doses of lipopolysaccharide (LPS) to mimic infection with gram‐negative bacteria. LPS treatment dose‐dependent increased contraction frequency of cardiac cell areas and calcium spikes and increased protein expression of α‐actinin. LPS treatment increased the expression of the macrophage marker CD68 and CD69, which is upregulated after activation on T cells, B cells and NK cells. LPS dose‐dependent increased protein expression of toll‐like receptor 4 (TLR4). Moreover, upregulation of NLR family pyrin domain containing 3 (NLRP3), IL‐1ß and cleaved caspase 1 was observed, indicating activation of inflammasome. In parallel, generation of reactive oxygen species (ROS), nitric oxide (NO), and expression of NOX1, NOX2, NOX4 and eNOS occurred. ROS generation, NOX2 expression and NO generation were downregulated by the TLR4 receptor antagonist TAK‐242 which abolished the LPS‐induced positive chronotropic effect of LPS. In conclusion, our data demonstrate that LPS induced a pro‐inflammatory cellular immune response in tissues derived from ES cells, recommending the in vitro model of embryoid bodies for inflammation research.

pathways involved in infection and inflammation. 4 Due to the ethical questionability of animal experiments and increased opposition of societies against animal experimentation, cellular models of infection were established and are profiting from technological developments in high-resolution fluorescence microscopy, including long-term single-cell time-lapse microscopy. 5 However, single cell and monolayer cell models are far away from the complexity of the infection process in organisms, which involves direct effects on different tissues and organs as well as reactions of the innate and acquired immune system. A new experimental approach is multicellular (organoid) cultures consisting of different cell types. 6  In cardiomyocytes derived from human induced pluripotent stem (iPS), cells LPS increased expression levels of pro-inflammatory and chemotactic cytokines and induced dysfunction of electrical activity. 10 LPS is the major component of the outer membrane of Gramnegative bacteria and is mainly acting via toll-like receptor 4 (TLR4), which is expressed in cells of the innate immune system, but also in other cell types including cardiac cells, smooth muscle cells 11 and the endothelium. 12 In the heart, TLR4 is an essential mediator of inflammatory and apoptosis processes and plays a pivotal role in the development of cardiovascular diseases. 13 Activation of TLR4 is initiated by binding of the serum lipopolysaccharide (LPS)-binding protein (LBP) to LPS to facilitate subsequent extraction of LPS monomers by CD14 and the delivery of LPS to the TLR4/MD-2 complex. TLR4 triggers two signalling pathways called the MyD88-dependent and the TRIF-dependent one after the adaptor proteins involved in their induction. 14 TLR4 is tied to the canonical activation of the NRLP3 inflammasome, which is a multimeric complex of NLRP3, ASC, NEK7 and pro-caspase 1, and promotes autoproteolysis and activation of caspase 1. The latter cleaves pro-IL-1β and pro-IL-18 to their secreted pro-inflammatory forms. Moreover, LPS can directly bind and activate caspase 11, which next stimulates the primed NLRP3 inflammasome and induces IL-1ß release and pyroptosis. 14 In the present study, the multicellular EB system derived from murine ES cells was used to investigate LPS-induced proinflammatory TLR-4-mediated signalling pathways. Our data show that LPS treatment of differentiated embryoid bodies exerted tachycardia in beating clusters of cardiac cells, raised ROS and NO, induced differentiation/proliferation of CD68 + and CD69 + cells, and activated the NLRP3 inflammasome. Thus, the embryoid model is a helpful tool to investigate processes of infection and inflammation in a disease-in-dish in vitro setting.

| Materials
Lipopolysaccharide from E. coli O111:B4 was purchased from Sigma-Aldrich, and TAK-242 was purchased from Cayman Chemical.
VAS2870 was a generous gift from Vasopharm.

| LPS and inhibitor treatment protocol
Embryoid bodies were removed from spinner flasks on Day 3 of differentiation and plated either onto 6 cm Petri dishes or gelatincoated glass cover slips in 24-wells, and further cultivated until Day 9 of cell culture, that is after completion of cardiac cell differentiation. Incubation with 0.1, 1 and 5 μg/mL LPS was performed from

| Western blot analysis
The western blot assays were carried out after washing embryoid reflects the relative amounts of protein as a ratio of each target protein band to the respective housekeeping protein.

| Statistical analysis
Data are given as mean values ± SD, with n denoting the number of experiments unless otherwise indicated. One-way anova for unpaired data and Student's t-test were applied as appropriate. A value of p ≤ 0.05 was considered significant.

| Expression of TLR4 in embryoid bodies upon LPS treatment
It is generally accepted that the inflammatory response of LPS is mediated via TLR4. 15 We therefore investigated, whether incubation with LPS would upregulate TLR4, and whether TLR4 is expressed in cardiomyocytes differentiated from ES cells. The data of the present study demonstrate that TLR4 was expressed in 10-day-old single cardiac and non-cardiac cells enzymatically dissociated from embryoid bodies ( Figure 1A). Cardiac cells were identified by doublelabelling with antibodies against sarcomeric α-actinin and cTnT ( Figure 1B). Moreover, TLR4 protein in embryoid bodies was dosedependent upregulated by LPS upon treatment from Day 9 to Day 15 of cell culture ( Figure 1C).

| Differentiation of leukocytes in embryoid bodies following treatment with LPS
The induction of inflammation in embryoid bodies may be associated with a stimulation of leukocyte differentiation, which has previously shown by us to occur within 15 days of cell culture. 16 In the present study, protein expression of the macrophage marker CD68 and CD69, which is expressed upon activation of T lymphocytes and natural killer (NK) cells, was investigated. Moreover, the number of of CD68 + and CD69 + cells enzymatically dissociated from embryoid bodies ( Figure 3E).

| Generation of ROS and expression of NOX enzymes upon LPS treatment
Inflammatory processes are associated with oxidative stress arising from ROS, which may generate by NOX enzymes and/or as side products of the mitochondrial respiratory chain. By use of the cy-

| Generation of NO upon treatment of embryoid bodies with LPS
Lipopolysaccharide has been reported to raise NO in previous studies. 17 To investigate, whether comparable effects would occur in embryoid bodies, the NO-specific fluorescence dye DAF-FM was used in microfluorometric experiments. Indeed, we observed that NO generation increased dose-dependent upon incubation with LPS ( Figure 5A).
Moreover, LPS treatment dose-dependent increased protein expression of NOS3 as well as phospho-NOS3 ( Figure 5B). The increase in NO generation elicited by LPS (5 μg/mL) was completely abolished in presence of TAK-242 (1 μM) ( Figure 5C and Figure S1), indicating activation of TLR4 signalling under these experimental conditions. Interestingly TAK-242 (1 μM) partially downregulated the intrinsic NO generation of embryoid bodies, which may suggest that inflammatory signalling cascades may take place during early embryogenesis. 18 pro-inflammatory immune response, may affect heart cell function and modulate LPS effects on the vitality and electrical properties of cardiomyocytes. Increase in heart rate is a known feature of septic shock, and prolonged elevation of the heart rate has been associated with poor outcome. 19 Tachycardia is arising from stimulation of ß1 adrenergic receptors through stimulation of the sympathetic nerve system. Consequently, sepsis-mediated tachycardia is clinically treated with ß-blockers, which significantly decreases the mortality of septic patients. 20 39 Notably, the data of the present study indicated that pre-treatment with TAK-242 significantly blunted the LPS-induced expression of NOX2 and inhibited ROS production.

| Activation of the NLRP3 inflammasome in embryoid bodies and ES cell-derived cardiac cells by LPS
Moreover, LPS-induced NO generation was completely abolished under these experimental conditions. Interestingly TAK-242 significantly depressed the endogenous NO production in the absence of LPS, which may indicate that TLR4 is involved in basal NO generation by embryoid bodies.
Lipopolysaccharide is eliciting an inflammatory response via the NLRP3 inflammasome. Indeed, the data of the present study demonstrated increased expression of NLRP3 in cardiac and non-cardiac cells, which suggests a general pro-inflammatory microenvironment within the tissue. NLRP3 promotes autoproteolysis and activation of caspase 1, which was confirmed in the present study. Moreover, activation of caspase 3 was observed, which remained, however, nonsignificant. Since caspase 1 cleaves pro-IL-1β protein, the number of IL-1ß + cells was assessed. Indeed, LPS treatment dose-dependent increased IL-1ß protein expression and the number of IL-1ß + cells.
Pre-treatment with TAK-242 totally abolished this effect.
Lipopolysaccharide-induced activation of the NLRP3 inflammasome has been above all investigated in laboratory animals and specific cell types, but so far not in complex tissues consisting of or-

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors confirm that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.